The broad objective of my research is to determine the molecular mechanism(s) responsible for regulation of crossbridges and contraction in vascular smooth muscle. Contractile stimuli were originally hypothesized to regulate smooth muscle contraction only via [Ca2+]i/calmodulin-dependent activation of myosin light chain kinase (MLCK). Activated MLCK is known to phosphorylate the 20 Kd light chain of myosin. Myosin light chain phosphorylation appears to be the primary determinant of force production. However, we found that myosin phosphorylation levels depend on both changes in [Ca2+]i and the stimulus employed. KC1 depolarization of intact smooth muscle tissues induced relatively larger increases in [Ca2+]i and relatively smaller increases in myosin phosphorylation than were observed with histamine stimulation (i.e. depolarization was associated with a lower [Ca2+]i sensitivity than was observed with histamine stimulation). There are four possible mechanisms that could explain changes in [Ca2+]i sensitivity: 1) the Ca2+-sensitivity of MLCK could be altered (e.g. by phosphorylation of MLCK), 2) myosin light chain phosphatase could be regulated, 3) [Ca2+]i estimates may be inaccurate, and/or 4 myosin could be phosphorylated by another kinase. The primary objective of this proposal is to determine the molecular mechanism(s) responsible for the alterations in [Ca2+]i sensitivity observed in intact vascular smooth muscle. The Specific Aims are to: 1) administratively deleted. 2) Test the hypothesis that aequorin accurately estimates myoplasmic [Ca2+] in intact tissues. We will measure myoplasmic [Ca2+] with both aequorin and Fura 2 in intact tissues. 3) Test the hypothesis that a G protein is involved in regulating [Ca2+]i sensitivity. We will inhibit G proteins with pertussis toxin or intracellularly loaded GDP-beta-S an evaluate the effect on [Ca2+]i and [Ca2+]i sensitivity.